Helicases are a class of enzymes that are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis. Many cellular processes (DNA replication, transcription, translation, recombination, DNA repair, ribosome biogenesis) involve the separation of nucleic acid strands. Helicases are often utilized to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases. They move incrementally along one nucleic acid strand of the duplex with a directionality and processivity specific to each particular enzyme. There are many helicases (14 confirmed in E. coli, 24 in human cells) resulting from the great variety of processes in which strand separation must be catalyzed.
Studies have shown that helicases do not merely wait passively for the fork to widen, but play an active role in forcing the fork to open, thus it is an active motor unwinding its substrate.
The current technologies available that attempt to examine helicase activity include: 1) ATPase activity assays, which are quantitative but do not provide a direct method of measuring unwinding activity; and 2) radioactive helicase assays, which are not quantitative but provide a direct measurement of unwinding (or helicase activity). However, both of these assays are very laborious. What is needed in the art are efficient, uniform methods and assays for determining helicase activity.